The physiological effects of opiates, including drugs of abuse such as heroin or prescription opiate medications, are mediated by G protein-coupled receptors (GPCRs). Activation of opioid GPCRs results in feedback mechanisms in which receptor number and signaling activity is regulated through endocytosis and sorting at the endosome. The current model describes three sorting operations for opioid GPCRs at the endosome: degrade at the lysosome, reinsert at the cell surface ('recycling'), or hold at the endosome ('retention'). The importance of this process in regulation of opioid function is underscored by the observation that the mu-opioid receptor (MOR) and the delta-opioid receptor DOR are sorted in diametrically opposed manners at the endosome: MOR recycles while DOR degrades. While the mechanisms and proteins involved in recycling and degradation of opioid receptors are becoming well understood, the role of the endosomal retention remains puzzling. The central paradox of endosomal retention comes from experimental data linking it to the sorting of DOR (which degrades) but also MOR (which recycles). These data raise the question of when and how endosomal retention occurs for opioid receptors in context of the general trafficking outcomes already described. Testing these questions has proven difficult, as for the last decade the only known component of the retention pathway was the protein GASP1 (GPCR-associated sorting protein 1), and its connection with the receptor is challenging to manipulate. An exciting recent development has been the identification of a new protein, named Beclin2, which forms a complex with GASP1 and is necessary for GASP1 function. Beclin2 is more amenable to experimental manipulation, opening the opportunity to directly test the role of endosomal retention in the regulation of opioid receptors. The hypothesis of this proposal, supported by preliminary data, is that the endosomal retention of opioid receptors functions as the initial sorting step for both the recycling and degrading pathways: Beclin2- GASP1 hold opioid GPCRs at endosomes until a handoff of the receptor mediates spatial separation and entry into the recycling or degrading pathways. This hypothesis will be rigorously tested using a combination of Determine the role of Beclin2 in sorting of defined manipulations (to the receptors and endosomal sorting machinery) with techniques including flow cytometry, microscopy, biochemistry, and mass spectrometry in primary neuronal cultures and non-neuronal recycling GPCRs; (2) Define the relative functional locations of Beclin2 and the endosomal recycling model cell lines. The specific aims of this proposal: (1) complex; (3) Establish the identity of the Beclin2 sorting complex. This proposal addresses opioid receptor regulation on the molecular level with the goal of better understanding the cellular response to opiate drug abuse and addiction.